Fusion expression, purification and activity characterization of alkaline phospholipase A_(2) from Lapemis hardwickii Gray

Phospholipase A_(2) (PLA_(2)) are the major components of snake venoms. The gene encoding alkaline PLA_(2) (PLA_(2)-9) from Lapemis hardwickii Gray was cloned into the 3^ end of the thioredoxin gene (trxA) in plasmid pETTRX to construct the pETRX-PLA_(2)-9 fusion expression vector. The TRX-PLA_(2)-9 was expressed as a soluble protein in E. coli induced by IPTG at 25deg C , and amounted to more than 20% of the total bacterial proteins. A fusion protein of PLA_(2)-9 with over 85% purity was obtained through two-step purification consisting of immobilized metal-chelate affinity chromatography and gel filtration. After cutting the fusion protein with enterokinase and further purification by cation-exchange chromatography on SP-Sepharose fast flow, the mature PLA_(2)-9 with 95% purity was obtained. The recombinant PLA_(2)-9 was detected by Western-blot analysis. The PLA_(2)-9 has the similar enzymatic activity to the natural PLA_(2) and has the cytotoxic activity on several tumor cell lines such as promyelocytic leukemia HL60. This is the first report on the sea snake PLA_(2) research.